We describe a strategy by which reactive binding of a weakly-bound, 'dynamically docked (DD)' complex without a known structure can be strengthened electrostatically through optimized placement of surface charges, and discuss its use in modulating complex formation between myoglobin (Mb) and cytochrome b 5 (b 5 ). The strategy employs paired Brownian Dynamics (BD) simulations, one which monitors overall binding, the other reactive binding, to examine [X → K] mutations on the surface of the partners, with a focus on single and multiple [D/E → K] charge reversal mutations. This procedure has been applied to the [Mb, b 5 ] complex, indicating mutations of Mb residues D44, D60 and E85 to be the most promising, with combinations of these showing a nonlinear enhancement of reactive binding. A novel method of displaying BD profiles shows that the 'hits' of b 5 on the surfaces of Mb(WT), Mb(D44K/D60K), and Mb(D44K/D60K/E85K) progressively coalesce into two 'clusters': a 'diffuse' cluster of hits that are distributed over the Mb surface and have negligible electrostatic binding energy; a 'reactive' cluster of hits with considerable stability that are localized near its heme edge, with short Fe-Fe distances favorable to electron transfer (ET). Thus binding and reactivity progressively become correlated by the mutations. This finding fits well with recent proposals that complex formation is a two-step process, proceeding through the formation of a weakly-bound encounter complex ('diffuse cluster') to a well-defined bound complex ('reactive cluster'). The design procedure has been tested through measurements of photoinitiated ET between the Zn-substituted forms of Mb(WT), Mb(D44K/D60K) and Mb(D44K/D60K/E85K) and Fe 3+ b 5 . Both mutants convert the complex from the DD regime exhibited by Mb(WT), in which the transient complex is in fast kinetic exchange with its partners, k off ≫ k et , to the slow-exchange regime, k et ≫ k off , and both mutants exhibit rapid intracomplex ET from the triplet excited state to Fe 3+ b 5 (rate constant, k et ~ 10 6 s −1 ). The affinity constants of the mutant Mbs cannot be derived through conventional analysis procedures because intracomplex singlet ET quenching causes the triplet-ground absorbance difference to progressively decrease during a titration, but this effect has been incorporated into a new procedure for computing binding constants. Most importantly, these measurements reveal the presence of fast photo-induced singlet ET across the protein-protein interface, 1 k et ≈ 2 × 10 8 s −1 .
We describe photo-initiated electron transfer (ET) from a suite of Zn-substituted myoglobin (1Mb) variants to cytochrome b5 (b5). An electrostatic interface redesign strategy has led to the introduction of positive charges in the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at `hotspot' residues (D44, D60, E85), augmented by the elimination of negative charges from Mb or b5 by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges: −5 < −qMbqb5 < 40. The binding affinity (Ka) increases a thousand-fold as −qMbqb5 increases through this range, and exhibits a surprisingly simple, exponential dependence on −qMbqb5. This is explained in terms of electrostatic interactions between a `charged reactive patch' (crp) on each partner's surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant, and (ii) encompasses a major fraction of the `reactive region' (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As −qMbqb5 increases, the complex undergoes a transition from fast to slow exchange dynamics on the triplet ET timescale, with a correlated progression in the rate constants for intracomplex (ket) and bimolecular (k2) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET, and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by applying it to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c2. Deviations from this model also are discussed.
Cognitive aids and evidence-based checklists are frequently utilized in complex situations across many disciplines and sectors. The purpose of such aids is not simply to provide instruction so as to fulfill a task, but rather to ensure that all contingencies related to the emergency are considered and accounted for and that the task at hand is completed fully, despite possible distractions. Furthermore, utilization of a checklist enhances communication to all team members by allowing all stakeholders to know and understand exactly what is occurring, what has been accomplished, and what remains to be done. Here we present a set of evidence-based critical event cognitive aids for neuroanesthesia emergencies developed by the Society for Neuroscience in Anesthesiology and Critical Care (SNACC) Education Committee.
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